Depletion method of blood plasma ascorbate

ABSTRACT

Artificial modulation of ascorbate level was investigated in mice capable of de novo synthesis of ascorbate. When mice were given exogenous ascorbate or its physiological precursor, L-gulono-γ-lactone, the plasma level of ascorbate was elevated substantially but immediately returned to the basal levels. Comparably, the administration of ascorbate oxidase caused a rapid disappearance of plasma ascorbate but followed by an immediate restoration of ascorbate. These results indicate the difficulties encountered in the modulation of ascorbate level in the animal. However, the circulation life of the exogenous ascorbate oxidase in the animal was successfully extended by chemical modification with methoxypolyethylene glycol. The modified enzyme retained a full activity and exerted a remarkably prolonged depletion of plasma ascorbate compared with the native enzyme. This study suggests that the chemically modified ascorbate oxidase should find many uses in the animal studies on ascorbate since it was found to deplete plasma ascorbate even in the ascorbate-synthesizing animal in the absence of dietary control. The enzyme should prove to be useful in tumor control because there are tumor systems in mice and man amenable to the manipulation of ascorbate level.

FIELD OF THE INVENTION

The present invention relates to a effective depletion method of bloodplasma ascorbate, by using ascorbate oxidase modified with biologicallyinert polymers.

DESCRIPTION OF THE PRIOR ART

<Desirability of L-ascorbic acid(LAA) in vivo manipulation forPre-Leukemia(Myelodysplastic Syndromes, MDS) and Acute MyeloidLeukemia(AML): overall perspectives and >

LAA has been shown to be an essential requirement for the growth ofmouse myeloma cells in an in vitro colony assay(1). When this findingwas applied to human myeloma, there appeared a similar effect althoughit was hard to test because human myeloma cell colonies do not grow wellin vitro(2). Human leukemia(AML) cell colonies grow well in ourdaily-feeding agar culture system(3), and an early study indicated thatLAA modulated growth of leukemic cells in approximately ½ of a smallnumber of patients(4). This has been confirmed in a large number ofpatients.

All these findings considered together indicate that leukemic cellssensitive to LAA in vitro may well be amenable to in vivo manipulationof LAA.

Most previous studies on the in vitro LAA effect were performed onleukemia (AML) cells. A more recent study indicates that thepre-leukemia, or myelodysplastic syndromes(MDS) generally considered tobe related to AML, has virtually an identical pattern in terms of LAAsensitivity, with 30% showing growth enhancement and 16% suppressionwith LAA(5).

<The recent lessons from a first patient subjected to LAA in vivomanipulation and multiple other patients from another independent group:the case for LAA supplementation>

The straightforward approach, exploiting above information clinically,to the patients in whom leukemic cell growth is enhanced by LAA wouldnaturally be in vivo depletion of LAA. A first patient was had onIRB(International Review Board)-approved protocol focusing on LAAdepletion and, indeed this patient appears to have had delay in relapseof leukemia by LAA depletion. However, the patient finally relapsedshowing the beginning of rapid rise in peripheral blast counts. Therethen was no choice but gradually increasing the dose of LAA in the hopeof increasing benefit, and that can suppress disease over 10 weeks.

<Inference from above rationales and experiences leading to cyclicapplication of bipolar approaches with extreme supplementation andextreme depletion>

It is compelling that high dose LAA indeed can suppress humanmalignancies. It is also believed that the case had benefit during LAAdepletion phase also by having prolonged period(12 wks) before frankrelapse, a consequence logically expected from the fact that patient'sleukemic cells growth was enhanced by LAA.

This kind of bipolar response is not unprecedented in treatment of humanmalignancies. In fact LAA is commonly known as an antioxidant but canbecome the other extreme, prooxidant at least in vitro(6,7), dependingfor example on metallic ion concentration around(8). This can be onepossible explanation for this bipolar activities. The extreme lack ofLAA may well be the situation of absence of antioxidant protection withendogenous and ubiquitous oxidants damaging cells unopposed. The extremehigh level of LAA with some other intra/extra cellular status(again suchas Fe++) may render it prooxidant leading to cell damage. Whatevermechanism might be, the dose response study with extension into highdose levels proves this bipolar inhibition on the leukemic cell growthof this first patient.

DISCLOSURE OF THE INVENTION

It is clear that ascorbate has effects on many physiological processesin humans [1]. Insufficient intake of ascorbate causes scurvy associatedwith decreased collagen synthesis. Ascorbate influences on woundhealing, gastric iron uptake, and many immunological and biochemicalreactions. Apparently, most, if not all, of ascorbate's functions arerelated to the reducing property of ascorbate keeping metal ions inreduced state. As a reductant, ascorbate can act either as anantioxidant or as a prooxidant in aqueous environments [2]. However,ascorbate is generally designated as an important antioxidant for humansbased on many in vitro findings [3–5]. Further, it is widely held thatascorbate may contribute to the prevention of pathological processesassociated with oxidative injury, but only circumstantial evidence isavailable [6, 7].

Ascorbate has also been implicated to affect the growth of animal cellsin vitro [8–11]. The effect appears as either stimulatory or inhibitorydepending on the cells. Although the precise mechanism for the growthmodulation by ascorbate is not known, the effect has been proven to bebiological rather than physicochemical since optical isomers ofascorbate or other redox compounds are without effect. Further, it isworthy to note that malignant cells are more sensitive than normalcounterparts to the ascorbate effects. We have shown the unusualascorbate sensitivity of malignant cells from patients with acutemyelogenous leukemia and myelodysplastic syndromes [12, 13].

There are compelling evidences that this in vitro effect will translateto in vivo situation ultimately resulting in tumor control. As explainedfully in the Description of the Prior Art, both extreme depletion andsupplementation will be beneficial to the control of cancer. Thereforethe depletion and supplementation periods will need to be cycled. Onestep further, for depletion to be maximumly effective priorsupplementation period will be desirable and vice versa. In thisApplication one of these 2 extremes, depletion, is the subject.

Animal studies are critical for the demonstration of the physiologicalrelevance of many proposed functions of ascorbate. For the experimentalpurpose, ascorbate level in animals needs to be modulated artificiallybut dietary controls are not so promising [14]. Ascorbate administeredin excess is readily excreted from the body and only minor fraction isequilibrated with body stores of ascorbate. Furthermore, such laboratoryanimals as mice are capable of endogenously synthesizing ascorbate, andby nature they are insensitive to dietary intake of ascorbate. Theimportance of animal studies on ascorbate modulation as related to tumorcontrol, and the usefulness of mice as laboratory animals prompted us toinvestigate new methods of ascorbate modulation in mice. Two oppositemethods are investigated in the present study, one for the elevation ofascorbate level and another for the selective depletion of ascorbate.

BRIEF DESCRIPTION OF THE ATTACHED DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1. Time-dependent changes of plasma ascorbate level in mice givensodium ascorbate (-o-) and L-gulono-γ-lactone (-•-). Drugs weredissolved in distilled water and administered by intraperitonealinjections, both at 5 mmole/kg. Blood was taken from each animaltime-sequentially for the assay of plasma ascorbate with HPLC-ECD, asdescribed in MATERIALS AND METHODS. Zero time control animals were given0.2 ml of normal saline. Data represent Mean±SD (n=3−5).

FIG. 2. Effects of exogenous ascorbate oxidase on plasma ascorbate levelin mice. Ascorbate oxidase in normal saline was intravenouslyadministered into BALB/c mice at various doses. Control animals weregiven 0.1 ml of normal saline. Plasma ascorbate level was assayed onehour after drug administration. Inset shows the time-dependent change ofplasma ascorbate level in mice given 400 units/kg of ascorbate oxidase.Data represent Mean±SD (n=3−5).

FIG. 3. SDS-PAGE of the modified ascorbate oxidase and the unmodified.Electrophoresis was performed on 6% gel and proteins were stained withCoomassie Blue. Lane I, ascorbate oxidase coupled to mPEG; lane II,ascorbate oxidase; lane III, standard molecular weight markers consistedof myosin H chain (200 kDa), phosphorylase (111 kDa), bovine serumalbumin (73 kDa) and ovalumin (47 kDa).

FIG. 4. Comparison of ascorbate depletion in mice given native ascorbateoxidase (-o-) and ascorbate oxidase coupled to mPEG (-•-). Drugs innormal saline were administered via intraperitoneal routes, both at 400units/kg. Control animals were given 0.1 ml of normal saline. Datarepresent Mean±SD (n=3−5).

MODES FOR CARRYING OUT THE INVENTION

Materials and Methods

Materials used in this invention are as follows.

Sodium ascorbate, L-gulono-γ-lactone, ascorbate oxidase (from Curcubitaspecies), methoxypolyethylene glycol (mPEG) activated with cyanuricchloride (average molecular weight 5000), borax, sodium citrate,mataphosphoric acid and dithiothreitol were purchased from SIGMA (St.Louis, Mo.). All other chemicals are of reagent grade.

Chemical modification of ascorbate oxidase is as follows.

Ascorbate oxidase (EC 1.10.3.3)) was modified with MPEG activated withcyanuric chloride, as in Jackson et al. [15]. Briefly describing, 4 mgof ascorbate oxidase was dissolved in 10 ml of ice-cold 0.1 M boraxbuffer (pH 9.2) and then 200 mg of activated mPEG was directly added tothe enzyme solution with stirring on ice. The reaction mixture was kepton ice for 60 min and dialyzed against normal saline at 4° C. across aSpectrapor-2 membrane (SPECTRUM. Houston, Tex.). The dialyzed protein,i.e. pegylated ascorbate oxidase, was used immediately or stored at −60°C. until used.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)was performed on 6% gel prepared by Laemmli's formulation [16] andproteins were stained with Coomassie Blue. Ascorbate oxidase activitywas assayed spectrophotometrically, as in Kim et al. [17].

Animals used in this invention are as follows.

Specific pathogen-free female BALB/c mice (body weight, ca. 25 g) wereused in the present study. Animals were obtained from Dae-Han LaboratoryAnimal Center (Daejeon, Korea) and cared for according to the ILARguidelines in the animal care facility of Samsung Biomedical ResearchCenter (Seoul, Korea). They were fed a diet from Jell Laboratory Chow(Seoul, Korea). Feed and water were offered ad libitum.

Extraction and assay of ascorbate are as follows.

The animal was anesthetized with diethyl ether vapour and blood wasobtained by choroidal venous plexus. About one ml of blood was collectedinto a microcentrifuge tube that contained 50 μl of 130 mM sodiumcitrate as an anticoagulant. The blood was centrifuged at 1000×g for 10min to obtain plasma, which was rapidly cooled on ice. Equal volume ofice-cold 10% metaphosphoric acid was added to the plasma and the mixturewas centrifuged at 10000×g for 10 min. The ascorbate content in thesupernatant was measured directly by HPLC method. An aliquot of thesample was treated with 100 mM dithiothreitol at neutral pH to reducedehydroascorbate to ascorbate, and then total ascorbate content wasmeasured. The content of dehydroascorbate was estimated from thedifference between the two measurements. In some cases, blood plasma wasdirectly ultrafiltrated by using a Microcon YM-10 centrifugal filterdevice (MILLIPORE, Bedford, Mass.), as described by Mendiratta et al.[5]. The ultrafiltrate was injected to HPLC either directly for theassay of ascorbate, or after incubation with dithiothreitol for theassay of total ascorbate (ascorbate plus dehydroascorbate).

Ascorbate was assayed by HPLC with the electrochemical detection, aspreviously described [11]. HPLC system (SHIMADZU, Tokyo, Japan)consisted of CBM-10A communication module, LC-10AD pump, SIL-10A autoinjector, CTO-10A column oven, and L-ECD-6A electrochemical detector.Separation was carried out on a Shim-pack CLC-ODS column (6 mm×15 cm)with a guard column of the same material (4 mm×1 cm). Mobile phase was50 mM potassium phosphate (pH 2.5) containing 1 mM EDTA, eluted at aflow rate of 1.0 ml/min. The potential of the glassy carbon workingelectrode was set at +0.6 V versus an Ag/AgCl reference electrode.

Data analysis is as follows. Statistical software package StatView 4.0(Abacus concepts, Berkeley, Calif.) was used for the data analysis.

Results and Discussion

Ascorbate is synthesized from glucose via the hexuronic acid pathway inascorbate synthesizing animals [18]. L-Gulono-γ-lactone is an immediateprecursor of ascorbate and its conversion into ascorbate is catalyzed byL-gulono-γ-lactone oxidase [19]. Human's inability to synthesizeascorbate is due to the lack of this enzyme [20]. As a physiologicalprecursor of ascorbate, L-gulono-γ-lactone might be useful for theelevation of ascorbate level in ascorbate-synthesizing animals includingmice. This notion was subjected to experimental scrutiny in the presentstudy (FIG. 1). When mice were given 5 mmole/kg of L-gulono-γ-lactone,there was a substantial increase in plasma level of ascorbate, inagreement with the capacity of the animal to synthesize ascorbate.However, the accumulated ascorbate rapidly diminished to normal levelswithin a few hours, similarly to the directly administered ascorbate (assodium salt, 5 mmole/kg), implicating that the animal has the capacityto keep ascorbate level from abnormal elevation. Plausible mechanismsmay include the renal excretion and metabolic consumption of excessiveascorbate [14, 21], and inhibition of L-gulono-γ-lactone oxidase byascorbate [22]. All these mechanisms would be beneficial for the life ofmice, but due to these mechanisms, we have hardly a chance toinvestigate the physiological consequences of ascorbate elevation in thelaboratory animal.

Ascorbate oxidase is an enzyme that catalyzes the oxygen-dependentoxidation of ascorbate to dehydroascorbate [23]. Due to the highsubstrate specificity and catalytic efficiency, the enzyme has widelybeen used in numerous in vitro studies on ascorbate, including selectivedetermination of ascorbate [23]. However, its compatibility with animalsystems is unknown. The enzyme was tested as a drug for the selectivedepletion of ascorbate in the present study. As shown in FIG. 2, theadministration of ascorbate oxidase into mice lowered plasma level ofascorbate in a dose-dependent manner. The ascorbate depletion was notobserved when the enzyme was heat-inactivated (data not shown). Theseresults implicate a competition in the blood between the activity ofexogenous ascorbate oxidase and the ascorbate maintenance mechanisms,such as ascorbate regeneration from dehydroascorbate by red blood cells[5, 25] and ascorbate release from the liver where de novo synthesis ofascorbate takes place [18]. The minimal dose of ascorbate oxidaseinducing a total clearance of plasma ascorbate appeared to be about 100units/kg equivalent to 1 μg protein/animal, indicating the effectivenessof ascorbate oxidase in the blood. Time-sequential monitoring of theascorbate level in mice given 400 units/kg of ascorbate oxidase,however, revealed a rapid restoration of plasma ascorbate, probablyassociated with short circulation life of the exogenous enzyme.

Enzymes can be chemically modified with retention of activities byattaching biologically inert polymers at sites other than the activesite. Such chemical modifications have been shown to prolong thecirculation life of the enzymes in animals, by eliminatingimmunogenicity of native proteins and/or reducing the renal excretion[26, 27]. mPEGs are very useful polymers in this respect and readily canbe attached to proteins by use of cyanuric chloride as a coupling agent[15]. Ascorbate oxidase could be chemically modified with cyanuricchloride-activated mPEG in the present study. A far excessive amount ofthe activated mPEG (1000 molecules per one enzyme molecule) was used toensure the reaction. The attachment of the polymers to the enzyme wasverified through SDS-PAGE (FIG. 3). The modified ascorbate oxidaseexhibited a very slow mobility on 6% gel compared with the unmodified.The former appeared as a broad band close to the stacking gel, whereasthe latter occurred as a sharp band with apparent molecular weight of 70kDa corresponding to the subunit [23]. Enzyme activity loss by themodification was very slight (less than 10%).

To assess the influence of chemical modification, mice were given eithernative ascorbate oxidase or the modified enzyme, both at 400 unit/kg,and plasma level of ascorbate was monitored time-sequentially. Drugswere administered via intraperitoneal injections that were foundeffective as intravenous routes. As shown in FIG. 4, the modified enzymeexerted a rapid clearance of plasma ascorbate as the native enzyme, inline with its retention of enzyme activity. Furthermore, the duration ofascorbate depletion was remarkably prolonged; the modified enzymedepleted for about 120 h compared with 3–4 h for the native enzyme.These results clearly demonstrate the benefit of chemical modificationto extend the circulation life and the action time of the exogenousenzyme in animals.

Ascorbate is just oxidized to dehydroascorbate by the activity ofascorbate oxidase. Nonetheless, any trace amount of dehydroascorbate wasnot detected in plasma during the ascorbate depletion (data not shown).At least two different methods of sample preparation employed in thepresent study gave the same results. The results are not surprising,however, since dehydroascorbate is known to be very unstable in plasma[28]. Although further studies are required, it is tentatively inferredthat the modified ascorbate oxidase may lead a rather comprehensivedepletion of plasma ascorbate including its oxidized form, by steadilyoxidizing available ascorbate to dehydroascorbate which decomposessubsequently.

In summary, the present paper describes two different approaches toattain the purpose of ascorbate modulation in mice capable of de novosynthesis of ascorbate. In the first trial, we tested the administrationof L-gulono-γ-lactone as a physiological precursor of ascorbate toelevate ascorbate level, but no significant merits over direct ascorbatesupplementation were observed. However, in the following trial, theexogenous ascorbate oxidase was found very effective for the depletionof plasma ascorbate. The catalytic efficiency of the enzyme appeared tocompete readily with the ascorbate synthesis and/or recycling mechanismsin animals. Employing a biologically inert polymer to the enzyme, wecould surmount the problem associated with the short circulation life ofthe exogenous enzyme. The chemical modification of enzyme did notrequire additional risks such as severe loss of enzyme activity ortoxicity to animals. The modified ascorbate oxidase should find numeroususes in vivo researches on ascorbate. Notably, it seems likely to beused in animal studies to demonstrate physiological relevancy of many invitro findings such as antioxidant actions of ascorbate [3–5]. The moststraight-forward application will be the growth retardation of malignantcells sensitive to ascorbate[8, 11–13], ultimately leading to thecontrol of malignancies.

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What is claimed is:
 1. A method for depleting ascorbate in blood plasmaextracted from a living body, by using ascorbate oxidase modified withbiologically inert polymers.
 2. The method for depleting ascorbate inblood plasma according to claim 1, wherein the biologically inertpolymers are attached at sites other than the active sites.
 3. Themethod for depleting ascorbate in blood plasma according to claim 1,wherein the biologically inert polymer is methoxypolyethylene glycol.